JP2012526174A - Novel acylated decasaccharides and their use as antithrombotic agents - Google Patents

Abstract

の十糖類（式中、Ａｃはアセチル基を表し、Ｒは式−ＯＨまたは−ＯＳＯ_３−の基を表す。）、およびこの調製の方法に関する。式（Ｉ）のオリゴ糖は、抗血栓剤として有用である。The present invention relates to a compound of formula (I) in any one form of acid form or pharmaceutically acceptable salt.

(Wherein Ac represents an acetyl group and R represents a group of the formula —OH or —OSO ₃ —), and a method for this preparation. The oligosaccharides of formula (I) are useful as antithrombotic agents.

Description

  The present invention relates to novel oligosaccharides, more specifically acylated decasaccharides, and their use as antithrombotic agents.

  Clotting is a defense mechanism that prevents excessive blood loss and microbial invasion. However, inadvertent blood clot formation and translocation can be detrimental, and antithrombotic drugs prevent blood clot formation and growth.

  Heparin and low molecular weight heparin (LMWH) are current standard therapies in the management of thromboembolic diseases. These anticoagulant activities are exerted through inhibition of coagulation factors, mainly activated factor X (FXa) and thrombin (factor IIa). This inhibitory effect is mediated by specific interactions between antithrombin (AT), a serine protease inhibitor of the serpin family, and heparin species.

  These agents are derived from animal sources and unfractionated heparin (UFH) is isolated from tissues such as lung or intestinal mucosa of porcine or bovine origin. LMWHs such as tinzaparin, ardeparin, dalteparin, enoxaparin, nadroparin or leviparin are obtained by enzymatic or chemical depolymerization of heparin.

  Heparin and LMWH are a complex mixture of molecules, a polymer that contains a number of sulfated polysaccharides, each consisting of a linear monosaccharide residue. Thus, the various polysaccharides present in heparin and LMWH differ in terms of their length as well as their chemical structure. Different degrees of sulfation and the presence of various 1 → 4 linked uronic acid and glucosamine disaccharide units give rise to complex overall structures (J. Med. Chem., 2003, 46, 2551-2554).

  Another class of antithrombotic drugs exists in synthetic oligosaccharides. Indeed, in the early 1980s it was concluded that the unique pentasaccharide domain in several heparin chains is the minimal sequence required to bind and activate antithrombin III (Biochimie, 2003, 85, 83-89). Fondaparinux sodium is a synthetic analog of this pentasaccharide obtained through over 60 steps of chemical synthesis. It is a selective inhibitor of factor Xa, commercialized for the prevention of thrombosis after orthopedic and abdominal surgery, prevention and treatment of deep vein thrombosis and pulmonary embolism, and the treatment of coronary artery disease is there.

  The structure-based design resulted in longer-acting analogs such as Idraparinux that displayed either selective factor Xa or dual inhibitory properties of Xa and IIa. A survey of improved pharmacodynamic profiles is more potent than heparin with respect to antithrombin activity, but without this side effect, such as clinical candidate SR1233781 (hexasaccharide compound) aimed at providing heparin mimetics This resulted in the synthesis of long oligosaccharides.

J. et al. Med. Chem. 2003, 46, 2551-2554 Biochimie, 2003, 85, 83-89

  The applicant has devised a new approach for the identification of new antithrombotic compounds. Starting from a mixture of oligosaccharides of LMWH, specific analysis and separation methods have made it possible to isolate oligosaccharides that provide advantageous antithrombotic properties that are useful in anticoagulant therapy.

  The oligosaccharides according to the invention have the formula (I)

（式中、Ａｃはアセチル基（すなわち式−ＣＯＣＨ_３の基）を表し、Ｒは式−ＯＨまたは−ＯＳＯ_３ ⁻の基を表し、波線はピラノース環の平面の下または上のいずれかに位置する結合を示す。）
に対応する。

Wherein Ac represents an acetyl group (ie, a group of formula —COCH ₃ ), R represents a group of formula —OH or —OSO ₃ ^— , and the wavy line is located either below or above the plane of the pyranose ring. Indicates the bond to be performed.)
Corresponding to

The oligosaccharide of formula (I) is a decasaccharide. The present invention includes a decasaccharide of formula (I) in either one of the acid form or the pharmaceutically acceptable salt form. In the acid form, the carboxylic acid (—COO ⁻ ) and sulfuric acid (—SO ₃ ^— ) functional groups take the form of —COOH and —SO ₃ H, respectively.

Oligosaccharides of formula (I), the term "pharmaceutically acceptable salts" refers to one or more -COO ^- and / or -SO ₃ ^- functional groups, ionic bonding to a pharmaceutically acceptable cation It is understood to mean the oligosaccharide that is being used. Preferred salts according to the invention are salts in which the cation is selected from alkali metal cations, and even more preferred are salts in which the cation is sodium (Na ⁺ ).

  More specifically, the present invention provides oligosaccharides of the following formulas (Ia) and (Ib):

に関する。

About.

The oligosaccharide of formula (Ia) corresponds to the decasaccharide of formula (I) wherein R represents a group of formula —OSO ₃ ^— , while the oligosaccharide of formula (Ib) has a R of group of formula —OH This corresponds to the decasaccharide of the formula (I) represented.

  According to the present invention, the compounds of formula (I) can be synthesized by high performance liquid chromatography (GPC), AT affinity chromatography and high performance liquid chromatography (including dynamic coating anion exchange chromatography and covalent anion exchange chromatography). Can be obtained from the LMWH product by using an orthogonal (mixed) separation method selected from (HPLC). According to the invention, these separation methods can be used in any possible combination of these.

Gel permeation chromatography can be performed on a column packed with Bio Gel P30 (Bio-Rad) circulated using NaClO ₄ . The selected fraction is desalted using techniques known in the art.

  AT affinity chromatography can be performed on columns packed with AT-Sepharose. The stationary phase is prepared by coupling human AT (1 g; Biomed) to CNBr-activated Sepharose 4B (Sigma). The methodology of Hook et al. (FEBS Letters, 1976, 66 (1), 90-3) is used to prepare AT columns that are eluted with a NaCl gradient.

  Dynamic coating anion exchange chromatography HPLC is achieved by using CTA-SAX chromatography (dynamic anion exchange chromatography with cetyltrimethylammonium). The CTA-SAX semi-preparative column was prepared on a column packed with Hypersil BDS C18 (5 μm) by Mourier, P .; A. J. et al. And Viskov, C.I. Coated as described by (Analytical Biochem., 2004, 332, 299-313). Column coating is performed on the analytical column by leaching a cetyltrimethylammonium hydrogensulfate solution in water / methanol. The mobile phase is a sodium methanesulfonate aqueous solution with a concentration varying between 0 and 2.5M. The pH is adjusted to 2.5 by addition of diluted methane sulfonic acid. Collected fractions are neutralized and desalted on Sephadex G-10 after pretreatment on Mega Bondelt C18 cartridge (Varian).

  Covalent anion exchange chromatography can be accomplished using anion exchange on an AS11 (Dionex) semi-preparative HPLC column. Any other anion exchange method may be performed using a column other than Dionex AS11.

  The final step for desalting the oligosaccharides thus obtained is carried out after neutralization of the collected fractions in order to recover the oligosaccharides of the invention in the desired salt form. Methods for desalting oligosaccharides are well known to those skilled in the art and include, for example, desalting on Sephadex G-10 columns.

  The following protocol describes in detail examples for the preparation of compounds (Ia) and (Ib) according to the invention in the form of the sodium salt. These protocols are included herein for purposes of illustration only and are not intended to limit the invention.

  In this example, compounds (Ia) and (Ib) are prepared by the following steps: gel permeation chromatography (GPC), then ATIII affinity chromatography, then CTA-SAX chromatography (dynamic coating anion exchange chromatography), It is then prepared from the starting LMWH product by performing covalent anion exchange chromatography.

Approximately 140 g of enoxaparin (commercially available from sanofi-avenis) is injected approximately 60 times for gel permeation on a column packed with Bio Gel P30 (200 cm × 5 cm) and NaClO ₄ 0. 100 at 100 ml / hour. Circulate with 2M. Each time lasts 24 hours. The decasaccharide fraction is collected and desalted on a column (100 cm × 7 cm) packed with Sephadex G-10 that is circulated with water to give about 18 g of the fraction.

  The total decasaccharide fraction is injected about 36 times into ATIII affinity chromatography, where about 500 mg is injected onto a 30 cm × 5 cm column. The low affinity part is eluted from the column with a 0.25 M NaCl solution buffered at pH 7.4 with 1 mM Tris at 6 ml / min. The high affinity decasaccharide fraction was extracted with a step gradient of NaCl (0.7, 1.15, 1.6, 2.05, 2.65 and 3M NaCl, pH 7.4 in 1 mM Tris-HCl). Elute. The NaCl gradient is monitored by conductivity and detection is in the 232 nm UV.

  The decasaccharide eluted in the affinity fraction with a conductivity of 85 to 120 mS / cm is collected, desalted on Sephadex G-10 and achieved in CTA-SAX semi-preparative chromatography (250 mm × 22 mm column) Used as starting material for the purification of Column coating is performed by leaching a 1 mM cetyltrimethylammonium hydrogensulfate solution in water / methanol (17: 8, v / v) for 4 hours. The mobile phase is a sodium methanesulfonate aqueous solution (Interchim) with a concentration varying between 0 and 2.5M. The pH is adjusted to 2.5 by addition of diluted methane sulfonic acid. Separation is achieved at 40 ° C. The salt concentration in the mobile phase is increased linearly from 0 to 2.5M over 60 minutes. The flow rate is 20 ml / min and 234 nm UV detection is used.

  Approximately 250 mg is injected separately in 5 portions, where 50 mg of the purified fraction is injected onto the column. The obtained fraction is neutralized and then controlled on a CTA-SAX analytical column (150 × 2.1 mm Hypersil BDS C18 (3 μm)). Fractions are collected and passed through a Mega Bondelut C18 cartridge (Varian) and desalted on Sephadex G-10.

After this step, the decasaccharides of formula (Ia) and (Ib) are obtained in the form of a mixture of insufficient purity. These final separations and purifications are accomplished on AS11 (Dionex) HPLC semi-preparative columns circulating in a NaClO ₄ gradient. Fractions containing decasaccharides (Ia) and (Ib) are controlled on a Dionex AS11 analytical column (250 × 2.1 mm) and desalted on Sephadex G-10. These proton NMR analyzes performed on a BRUCKER instrument (600 MHz) are as follows.

Decasaccharide (Ia):
NMR ¹ H, in D ₂ O (δ, ppm): 2.05 (3H, s), 3.29 (2H, m,), 3.40 (1H, t, 6 Hz), 3.46 (1H, dd, 8 and 2 Hz), 3.65-4.55 (31 H, m), 4.61 (1 H, d, 6 Hz), 4.80 (3 H, m), 5.06 (1 H, d, 2 Hz) , 5.09 (1H, d, 2Hz), 5.17 (1H, d, 6Hz), 5.27 (1H, d, 2Hz), 5.36 (2H, m), 5.43 (1H, d , 2 Hz), 5.45 (1 H, d, 2 Hz), 5.53 (1 H, d, 2 Hz), 5.81 (1 H, d, 4 Hz).

Decasaccharide (Ib):
NMR ¹ H, in D ₂ O (δ, ppm): 2.04 (6H, s), 3.27 (2H, m), 3.42 (2H, m), 3.60-4.50 (40H) , M), 4.55 (1H, d, 6 Hz), 4.62 (1H, s), 4.80 (3H, m), 4.97 (1H, s), 5.02 (1H, s) 5.20 (1 H, d, 2 Hz), 5.35 (1 H, d, 2 Hz), 5.38 (2 H, m), 5.50 (2 H, m), 5.53 (1 H, s), 5.98 (1H, d, 4 Hz).

  The oligosaccharides of the present invention, after pharmacological studies, have shown this antithrombotic property and this value as a therapeutically active substance.

Anti-FXa activity in plasma:
The ability of the oligosaccharide (I) sodium salt to promote AT-mediated FXa inhibition was analyzed at near physiological conditions. Measurement of anti-FXa activity was carried out according to the manufacturer's recommendations as a competitive colorimetric assay STA®-Rotachrom® heparin (Diagonostica Stago) operated on a STA®-R analyzer (Diagnostic Stago Inc.). Inc.). Bovine FXa (Diagnostic Stago Inc.) was used. Fondaparinux obtained from a commercial source marketed by GlaxoSmithKline was the standard. This was spiked in normal pooled human plasma (Hyphen) with increasing concentrations (0.0218-0.0460-0.0872-0.1740-0.3490-0.4650 μmol / L). A dose response line was shown. The oligosaccharides and fondaparinux of the present invention were tested at 6 concentrations ranging from 0.0218 to 0.4650 μM. The concentration of AT in the plasma environment was 2.25 μM. The measured absolute anti-Xa activity of the purified oligosaccharide was expressed in IU / ml according to the European Pharmacopeia 6.0 (01/2008: 0828). Relative anti-FXa activity was calculated from the ratio of absolute activity to the activity of fondaparinux.

  In this test, the oligosaccharides (Ia) and (Ib) of the present invention exhibit absolute anti-FXa activity of 1.13 and 1.14 IU / ml, respectively. This relative anti-Xa activity compared to fondaparinux is 1.40 and 1.37 times, respectively.

  Thus, the oligosaccharides of formula (I) according to the present invention exhibit high antithrombotic properties. This oligosaccharide may be useful in the preparation of drugs, particularly antithrombotic drugs. Accordingly, another object of the present invention is a drug comprising this addition salt with an oligosaccharide of formula (I) or a pharmaceutically acceptable salt.

  Such drugs can be used to treat thrombosis, including therapy, especially venous thrombosis (eg, in post-operative surgery patients, cancer patients, or internal medicine patients with limited mobility) and acute arterial thrombotic events. And prevention, especially in the case of myocardial infarction.

  Another object of the present invention is a pharmaceutical composition comprising the oligosaccharide of formula (I) according to the present invention as an active ingredient. Such a pharmaceutical composition comprises an effective amount of an oligosaccharide of formula (I) according to the invention, or this addition salt with a pharmaceutically acceptable salt, and at least one pharmaceutically acceptable additive. Including. The additive is selected from conventional additives known to those skilled in the art according to the desired pharmaceutical form and administration route.

  The pharmaceutical composition according to the invention, whether in addition to the oligosaccharide of formula (I), is a synthetic compound (obtained by chemical stepwise synthesis starting from a suitable monosaccharide or oligosaccharide basic unit) Whether it is a compound isolated from a heparin or LMWH source, it can contain at least one other active ingredient selected from antithrombotic oligosaccharides.

  In a pharmaceutical composition according to the invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, transdermal or rectal administration, a compound of formula (I ) Active ingredient or a salt thereof can be administered to animals and humans as a unit dosage form by blending with usual pharmaceutical additives for the prevention or treatment of the above-mentioned pathological conditions.

  Suitable unit dosage forms include tablets, hard or soft gelatin capsules, powders, granules, and oral dosage forms such as oral solutions or suspensions, sublingual, buccal, intratracheal, intraocular, intranasal dosage forms by inhalation, Includes topical, transdermal, subcutaneous, intramuscular or intravenous dosage forms, rectal dosage forms and implants. For topical application, the compounds of the invention can be used as creams, gels, ointments or lotions.

  The invention also according to another of this aspect comprises the administration to a patient of an effective amount of a salt of the oligosaccharide of formula (I) according to the invention, or a pharmaceutically acceptable salt thereof, It relates to a method for the treatment and prevention of medical conditions.

Claims (14)

Oligosaccharides of formula (I) in either one of the acid form or the pharmaceutically acceptable salt form:

(In the formula, Ac represents an acetyl group, R represents a group of the formula —OH or —OSO ₃ ^— , and the wavy line represents a bond located either below or above the plane of the pyranose ring). R has the formula -OSO ₃ ^- represents a group of oligosaccharide of claim 1.   The oligosaccharide according to claim 1, wherein R represents a group of formula -OH.   The oligosaccharide according to any of claims 1 to 3, in the form of a sodium salt.   Starting by performing gel permeation chromatography (GPC), AT affinity chromatography, dynamic coating anion exchange chromatography (CTA-SAX) and covalent anion exchange chromatography in any possible combination of these methods 5. A method for the preparation of an oligosaccharide according to any of claims 1 to 4, comprising a step for separating the oligosaccharide from an LMWH product. The following steps:
Comprising gel permeation chromatography (GPC), then -AT affinity chromatography, then -dynamic coating anion exchange chromatography (CTA-SAX), and then -covalent anion exchange chromatography, Item 6. The method according to Item 5.   7. A method according to claim 5 or claim 6, characterized in that anion exchange chromatography is carried out on a Dionex AS11 HPLC column.   8. A process according to any one of claims 5 to 7, characterized in that the starting LMWH product is enoxaparin.   5. An oligosaccharide of formula (I) according to any of claims 1 to 4 for use as a drug, or this addition salt with a pharmaceutically acceptable salt.   A medicament comprising an oligosaccharide of formula (I) according to any of claims 1 to 4, or this addition salt with a pharmaceutically acceptable salt, and at least one pharmaceutically acceptable additive. Composition.   The pharmaceutical composition according to claim 10, further comprising at least one other active ingredient selected from antithrombotic oligosaccharides.   Use of an oligosaccharide of formula (I) according to any of claims 1 to 4 or a pharmaceutically acceptable salt thereof for the preparation of a medicament intended for the treatment and prevention of thrombosis .   Use according to claim 12, for the preparation of a medicament intended for the treatment and prevention of venous thrombosis and acute thrombotic events.   A method for the treatment and prevention of thrombosis comprising the administration to a patient of an effective amount of this addition salt with an oligosaccharide of formula (I) according to any of claims 1 to 4 or a pharmaceutically acceptable salt. Way for.